Isophorone dye derivative LYC-CF3 as well as synthesis method and application thereof

Abstract

The invention provides an isophorone dye derivative LYC-CF3 as well as a synthesis method and application thereof. The specific name of the isophorone dye derivative is (E)-4-(2-(3-(dicyanomethylene)-5, 5-dimethylcyclohex-1-ene-1-yl) vinyl)-2-(trifluoromethyl) phenyl trifluoromethanesulfonate, and the structural formula of the isophorone dye derivative is shown in the description. The invention also provides a synthesis method of the derivative LYC-CF3, and a fluorescence imaging experiment of the derivative LYC-CF3 on O2 <-> in PC-12 cells. Through verification of a fluorescence spectrum and a laser confocal microscope, the derivative LYC-CF3 has quick response to O2 <-> in an in-vitro test and cells and relatively remarkable fluorescence imaging capability. Therefore, LYC-CF3 can be used as a fluorescent probe for in-vitro detection of O2 <-> and detection of O2 <-> in cells.

Description

Technical Field

[0001] This invention relates to isophorone dye derivatives and superoxide anion fluorescent probes, specifically to an isophorone dye derivative, its synthesis method, and its application as a superoxide anion fluorescent probe. Background Technology

[0002] Superoxide anions, as a crucial member of the reactive oxygen species (ROS) cluster, play a key role in physiological processes such as cell signal transduction and immune defense. However, under oxidative stress, excessive production of superoxide anions can react with biomolecules, leading to lipid peroxidation, protein denaturation, and DNA damage, which are closely related to various pathological processes such as atherosclerosis, neurodegenerative diseases, and cancer. However, O2... •⁻ The detection faces two major technical bottlenecks: First, superoxide anion (O2) •⁻ It is chemically reactive but has low selectivity. It readily transforms into other reactive oxygen species (such as H₂O₂ and •OH); secondly, under physiological conditions, O₂... •⁻ Its steady-state concentration is extremely low, and its lifetime is extremely short. These characteristics make it suitable for developing highly selective and highly sensitive O2. •⁻ Developing detection tools has become an extremely challenging task.

[0003] Traditional methods for superoxide anion detection, such as electron paramagnetic resonance, chemiluminescence, and high-performance liquid chromatography, typically suffer from limitations such as complex operation, high sample destructiveness, and low spatiotemporal resolution, making them unsuitable for real-time dynamic monitoring at the live cell level. In contrast, fluorescence imaging technology, with its advantages of ease of operation, high sensitivity, non-invasiveness, and excellent spatiotemporal resolution, has become a powerful visualization tool in life science research.

[0004] Designing and synthesizing fluorescent probes that can specifically recognize superoxide anions has become a research hotspot. Existing fluorescent probes are mainly based on specific response mechanisms and usually consist of three parts: a fluorescent reporter group, a linker unit, and a recognition receptor that specifically responds to superoxide anions. However, many probes in the existing technology still face the following challenges: (1) Insufficient selectivity: Many probes also respond to other ROS (such as hydrogen peroxide, hypochlorous acid, hydroxyl radicals, etc.) or reactive nitrogen species (RNS), resulting in signal interference; (2) Limited response sensitivity: Within the physiological concentration range, the fluorescence enhancement factor or signal change is not significant enough, making it difficult to detect small concentration fluctuations; (3) Poor biocompatibility: Some probes have poor water solubility, high cytotoxicity, or difficulty in crossing biological barriers (such as the blood-brain barrier), limiting their application in complex biological systems. Therefore, developing a novel superoxide anion fluorescent probe with high selectivity, high sensitivity, good biocompatibility, and excellent optical performance to meet the needs of real-time and accurate imaging at the living cell and in vitro levels remains a technical problem that urgently needs to be solved in this field. Based on this, the present invention uses trifluorosulfonic acid groups as O2. •⁻ The reactivity of the trifluorosulfonic acid group was modulated by innovatively introducing –CF3 at the ortho position of the isophorone dye derivative's reaction site. The introduction of –CF3 significantly improved the optical properties of LYC-CF3, including sensitivity, selectivity, and signal-to-background ratio. Finally, due to the presence of isophorone fluorescent dyes, this isophorone dye derivative, LYC-CF3, exhibits strong lipophilicity and biocompatibility. Summary of the Invention

[0005] The purpose of this invention is to provide a method for synthesizing the isophorone dye derivative LYC-CF3, and to demonstrate the effectiveness of this derivative LYC-CF3 as a fluorescent probe against oxidative stress (O2) in vitro and in cells. •⁻ Applications of detection.

[0006] This invention provides an isophorone dye derivative, LYC-CF3, specifically named (E)-4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)-2-(trifluoromethyl)phenyl trifluoromethanesulfonate. Its structural formula is:

[0007] LYC-CF3 The method for synthesizing LYC-CF3 provided by this invention includes the following steps: 1) Add 5 equivalents of isophorone and 6 equivalents of malononitrile to a 100 mL round-bottom flask, then add 50 mL of anhydrous ethanol and 200 μL of piperidine and reflux at 85 °C overnight; concentrate the reaction solution under vacuum, and purify the reaction residue by silica gel column chromatography (PE / DCM) to obtain a yellowish-white solid compound 1. 2) Add 1 equivalent of compound 1 and 4-hydroxy-3-(trifluoromethyl)benzaldehyde to a 100 mL round-bottom flask, and add piperidine as a catalyst using anhydrous ethanol as the reaction solvent; after reacting overnight, concentrate the reaction solution under vacuum, and purify the reaction residue by silica gel column chromatography (PE / DCM) to obtain red solid compound 2. 3) Under ice bath conditions, add 1 equivalent of compound 2 to a 50 mL round-bottom flask, using anhydrous dichloromethane as the reaction solvent, and simultaneously add 1.5 equivalents of triethylamine. Stir for 15 min, then add 1.2 equivalents of trifluoromethanesulfonyl chloride and react for 5 h. After the reaction is complete, concentrate the reaction solution under vacuum, and purify the reaction residue by silica gel column chromatography (PE / DCM) to obtain a pale yellow solid compound 3, which is the isophorone dye derivative LYC-CF3.

[0008] The above-described synthesis is based on an isophorone dye derivative, LYC-CF3, as... - Fluorescent probes are used for rapid and specific qualitative detection of superoxide anions in vitro, and for the efficient uptake of the derivative LYC-CF3 in PC-12 cells and the detection of cellular oxidative stress. This includes: (1) Prepare a 2 mM LYC-CF3 stock solution; prepare a 1 mM superoxide anion stock solution; (2) Take 10 μL of LYC-CF3 and add it to 2 mL of the test system in a cuvette, then add the prepared O2. •⁻ The fluorescence intensity at 655 nm was detected using a fluorescence spectrophotometer with 525 nm UV-Vis absorption as the excitation source to assess the response of LYC-CF3.

[0009] (3) Preparation of 1. Blank. 2. Asp. 3. Pro. 4. Glu. 5. Gly. 6. GABA. 7. Thr. 8. His. 9. Arg. 10. Lys. 11. Ser. 12. Leu. 13. Val. 14. Trp. 15. Phe. 16. KCl. 17. CaCl2. 18. CuSO4. 19. MgCl2. 20. NaCl. 21. Na2CO3. 22. NaHCO3. 23. Ca(ClO)2. 24. H2O2. 25. Na2S. 26. NaHSO3. 27. NaHS. 28. Hcy. 29. GSH. 30. Cys. 31. O2 •⁻ Amino acids (500 μM), salt ions (500 μM), reactive oxygen species (200 μM), reactive sulfur (100 μM), and physiologically appropriate concentrations of Cys, Hcy, and GSH (200 μM, 15 μM, and 2 mM, respectively) were added to 10 μL of LYC-CF3 stock solution, 2.0 mL of the test system, and a cuvette. A blank group and other interfering substances (amino acids 500 μM, salt ions 500 μM, reactive oxygen species 200 μM, reactive sulfur 100 μM, and physiologically appropriate concentrations of Cys, Hcy, and GSH 200 μM, 15 μM, and 2 mM, respectively) were added to each. The fluorescence intensity at 655 nm was measured using a fluorescence spectrophotometer with 525 nm UV-Vis absorption as the excitation source to detect the effect of LYC-CF3 on O2. •⁻ The selectivity.

[0010] (4) In the laser confocal experiment, PC-12 cells were subjected to endogenous and exogenous O2. •⁻ Test, first on endogenous O2 •⁻For testing, 10 μL of LYC-CF3 was added to 2 mL of DMEM, shaken well, and then poured into a culture dish containing PC-12 cells. The cells were incubated for 10 minutes, washed three times with PBS, and placed under a fluorescence confocal microscope. The fluorescence intensity of the green and red channels in the 590-700 nm wavelength range was detected using a 525 nm excitation source; weak red fluorescence was observed. Then, PC-12 cells were incubated with 50 μM 2-ME for 30 minutes, washed three times with PBS, and placed under a fluorescence confocal microscope. The fluorescence intensity of the red channel in the 590-700 nm wavelength range was detected using a 525 nm excitation source; the red fluorescence intensity increased. PC-12 cells were co-incubated with 50 μM 2-ME and 20 μM Tiron for 30 minutes, washed three times with PBS, and placed under a fluorescence confocal microscope. The fluorescence intensity of the red channel in the 590-700 nm wavelength range was detected using a 525 nm excitation source; the red channel fluorescence decreased. Finally, PC-12 cells were incubated with 50 mM H2O2 and Glu for 30 min, then washed three times with PBS solution and placed on a fluorescence confocal microscope with 525 nm as the excitation source to detect the fluorescence intensity of the green and red channels in the 590-700 nm wavelength range. The fluorescence of the red channel was enhanced.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention is based on isophorone dyes, which offer a simple detection method and represent a significant breakthrough in fluorescence wavelength and selectivity. O2 can be detected using fluorescence spectroscopy. •⁻ In vitro qualitative and quantitative detection of O2 was performed. The effects of LYC-CF3 on O2 were verified by spectroscopy and confocal microscopy. •⁻ It has strong fluorescence imaging capabilities.

[0012] 2. O2 was controlled using LYC-CF3. •⁻ Rapid response and O2 in PC-12 cells •⁻ It exhibits a relatively significant fluorescence imaging capability. Attached Figure Description

[0013] Figure 1 1H NMR spectrum of LYC-CF3 Figure 2 LYC-CF3 C NMR spectrum Figure 3 Mass spectrum of LYC-CF3 Figure 4 UV and fluorescence response diagrams of LYC-CF3 Figure 5 Selectivity test chart of LYC-CF3 Figure 6LYC-CF3 affects the endogenous and exogenous O2 in PC-12 cells. •⁻ Laser confocal test diagram Detailed Implementation

[0014] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments. Example 1

[0015]

[0016] 1) Add 690 mg (5 mmol) of isophorone and 384 mg (6 mmol) of malononitrile to a 100 mL round-bottom flask. Then add 50 mL of anhydrous ethanol and 200 μL of piperidine to the flask and reflux overnight at 85 °C. Concentrate the reaction solvent under vacuum and purify the reaction residue by silica gel column chromatography (PE / DCM) to obtain a yellowish-white solid compound 1 (yield 80%). 2) 186 mg (1 mmol) of compound 1 and 228 mg (1.2 mmol) of 4-hydroxy-3-(trifluoromethyl)benzaldehyde were added to a 100 mL round-bottom flask. Anhydrous ethanol was used as the reaction solvent and a catalytic amount of piperidine was added. The reaction was carried out overnight. The reaction solution was concentrated under vacuum, and the residue was purified by silica gel column chromatography (PE / DCM) to obtain red solid compound 2 (yield 60%). 3) Add 358 mg (1 mmol) equivalent of compound 2 to a 50 mL round-bottom flask under ice bath conditions, using anhydrous dichloromethane as the reaction solvent, and simultaneously add 151 mg (1.5 mmol) of triethylamine. Stir for 15 min, then add 200.4 mg (1.2 mmol) of trifluoromethanesulfonyl chloride and react for 5 h. After the reaction is complete, concentrate the reaction solution under vacuum, and purify the residue by silica gel column chromatography (PE / DCM) to obtain a pale yellow solid compound 3, which is the isophorone dye derivative LYC-CF3 (yield 40%). 1 H NMR(600 MHz, DMSO-d6) δ 8.31 (s, 1H), 8.15 (d, J = 8.7 Hz, 1H), 7.76 (d, J = 8.7Hz, 1H), 7.66 (d, J = 16.2 Hz, 1H), 7.40 (d, J = 16.2 Hz, 1H), 6.98 (s, 1H), 2.64 (s, 2H), 2.54 (s, 2H), 1.02 (s, 6H). 13C NMR (151 MHz, DMSO-d6) δ 170.63,155.04, 145.06, 137.90, 134.46, 133.95, 133.61, 127.41, 127.38, 127.35,127.32, 124.92, 124.02, 123.39, 122.52, 122.30, 121.58, 119.42, 117.30,114.01, 113.25, 78.51, 42.66, 38.56, 32.13, 27.88. ESI-MS: [M] - Calcd. For489.0713, Found 489.07043.( Figure 1 , Figure 2 , Figure 3 ) Example 2

[0017] Add 10 μL of LYC-CF3 to 2 mL of the test system in a cuvette, then add the prepared O2. •⁻ The fluorescence intensity at 655 nm was measured using a fluorescence spectrophotometer with 525 nm UV-Vis absorption as the excitation source to detect the response of LYC-CF3. A significant emission peak was found at 655 nm for LYC-CF3. Figure 4 ) Example 3

[0018] (1) Preparation of 1. Blank. 2. Asp. 3. Pro. 4. Glu. 5. Gly. 6. GABA. 7. Thr. 8. His. 9. Arg. 10. Lys. 11. Ser. 12. Leu. 13. Val. 14. Trp. 15. Phe. 16. KCl. 17. CaCl2. 18. CuSO4. 19. MgCl2. 20. NaCl. 21. Na2CO3. 22. NaHCO3. 23. Ca(ClO)2. 24. H2O2. 25. Na2S. 26. NaHSO3. 27. NaHS. 28. Hcy. 29. GSH. 30. Cys. 31. O2 •⁻Amino acids (500 μM), salt ions (500 μM), reactive oxygen species (ROS) (200 μM), reactive sulfur (100 μM), and physiologically balanced Cys, Hcy, and GSH (200 μM, 15 μM, and 2 mM, respectively) were added to 10 μL of LYC-CF3 stock solution, 2.0 mL of the test system, and a cuvette. A blank group and other interfering substances (amino acids 500 μM, salt ions 500 μM, ROS 200 μM, RSO 100 μM, and physiologically balanced Cys, Hcy, and GSH (200 μM, 15 μM, and 2 mM, respectively) were added to each. The fluorescence intensity at 655 nm was measured using a fluorescence spectrophotometer with 525 nm UV-Vis absorption as the excitation source to detect the effect of LYC-CF3 on O2. •⁻ The selectivity. Figure 5 ) (2) In the laser confocal experiment, PC-12 cells were subjected to endogenous and exogenous O2. •⁻ Test, first on endogenous O2 •⁻ For testing, 10 μL of LYC-CF3 was added to 2 mL of DMEM, shaken well, and then poured into a culture dish containing PC-12 cells. The cells were incubated for 10 minutes, washed three times with PBS, and placed under a fluorescence confocal microscope. The fluorescence intensity of the green and red channels in the 590-700 nm wavelength range was detected using a 525 nm excitation source; weak red fluorescence was observed. Then, PC-12 cells were incubated with 50 μM 2-ME for 30 minutes, washed three times with PBS, and placed under a fluorescence confocal microscope. The fluorescence intensity of the red channel in the 590-700 nm wavelength range was detected using a 525 nm excitation source; the red fluorescence intensity increased. PC-12 cells were co-incubated with 50 μM 2-ME and 20 μM Tiron for 30 minutes, washed three times with PBS, and placed under a fluorescence confocal microscope. The fluorescence intensity of the red channel in the 590-700 nm wavelength range was detected using a 525 nm excitation source; the red channel fluorescence decreased. Finally, PC-12 cells were incubated with 50 mM H2O2 and Glu for 30 min, then washed three times with PBS solution and placed under a fluorescence confocal microscope with 525 nm excitation light source to detect the fluorescence intensity of the green and red channels in the 590-700 nm wavelength range. The red channel fluorescence was enhanced. Figure 6 ) The above experimental results indicate that LYC-CF3 is a reliable indicator for detecting O2 both in vitro and intracellularly. •⁻ A good tool.

Claims

1. An isophorone-based dye derivative LYC-CF3, characterized by The structural formula is: 。 2. The method for synthesizing the isophorone dye derivative LYC-CF3 as described in claim 1, characterized in that, The method comprises the following steps: 1) 5 equivalents of isophorone and 6 equivalents of malononitrile are added into a 100 mL round-bottom flask, 50 mL of anhydrous ethanol is added into the flask, 200 μL of piperidine is added, and the mixture is refluxed at 85°C overnight; The reaction solution is concentrated under vacuum, and the reaction residue is purified by a silica gel column (PE / DCM) to obtain yellow-white solid compound 1; 2) 1 equivalent of compound 1 and 4-hydroxy-3-(trifluoromethyl) benzaldehyde are added into a 100 mL round-bottom flask, anhydrous ethanol is used as a reaction solvent, and a catalytic amount of piperidine is added; after the reaction is completed overnight, the reaction solution is concentrated under vacuum, and the reaction residue is purified by a silica gel column (PE / DCM) to obtain red solid compound 2; 3) 1 equivalent of compound 2 is added into a 50 mL round-bottom flask under ice bath conditions, anhydrous dichloromethane is used as a reaction solvent, 1.5 equivalents of triethylamine is added, and the mixture is stirred for 15 min, then 1.2 equivalents of trifluoromethylsulfonyl chloride is added, and the mixture is reacted for 5 h; after the reaction is completed, the reaction solution is concentrated under vacuum, and the reaction residue is purified by a silica gel column (PE / DCM) to obtain yellowish solid compound 3, which is an isophorone dye derivative LYC-CF3.

3. Isophorone dye derivative LYC-CF3 according to claim 1 as fluorescent probe for the fluorescence spectroscopic detection of O2 •– .

4. Use of the isophorone dye derivative LYC-CF3 according to claim 1 for the preparation of a fluorescent probe for the detection of O2 in cells. •– 5. The use according to claim 4, wherein the probe is used for the detection of O2 in mitochondria.

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